Stem-loop Real-time PCR Research Articles

Identification and characterization of microRNAs in Clonorchis sinensis of human health significance

BackgroundClonorchis sinensis is a zoonotic parasite causing clonorchiasis-associated human disease such as biliary calculi, cholecystitis, liver cirrhosis, and it is currently classified as carcinogenic to humans for cholangiocarcinoma. MicroRNAs (miRNAs) are non-coding, regulating small RNA molecules which are essential for the complex life cycles of parasites and are involved in parasitic infections. To identify and characterize miRNAs expressed in adult C. sinensis residing chronically in the biliary tract, we developed an integrative approach combining deep sequencing and bioinformatic predictions with stem-loop real-time PCR analysis.ResultsHere we report the use of this approach to identify and clone 6 new and 62,512 conserved C. sinensis miRNAs which belonged to 284 families. There was strong bias on families, family members and sequence nucleotides in C. sinensis. Uracil was the dominant nucleotide, particularly at positions 1, 14 and 22, which were located approximately at the beginning, middle and end of conserved miRNAs. There was no significant "seed region" at the first and ninth positions which were commonly found in human, animals and plants. Categorization of conserved miRNAs indicated that miRNAs of C. sinensis were still innovated and concentrated along three branches of the phylogenetic tree leading to bilaterians, insects and coelomates. There were two miRNA strategies in C. sinensis for its parasitic life: keeping a large category of miRNA families of different animals and keeping stringent conserved seed regions with high active innovation in other places of miRNAs mainly in the middle and the end, which were perfect for the parasite to perform its complex life style and for host changes.ConclusionsThe present study represented the first large scale characterization of C. sinensis miRNAs, which have implications for understanding the complex biology of this zoonotic parasite, as well as miRNA studies of other related species such as Opisthorchis viverrini and Opisthorchis felineus of human and animal health significance.

Suppression of p21 by c-Myc through members of miR-17 family at the post-transcriptional level

The miR-17 family, composed of miR-17-5p, miR-20a/b, miR-106a/b and miR-93, has been demonstrated to take part in critical pathways that regulate cellular life and death decisions during normal development and in malignancy. Previous studies have shown that the expression of miR-17 family members has close relationship with the expression of c-Myc. Another study has reported p21 is a direct target of miR-17 family and their silencing of p21 contributes to tumor cell proliferation in part. Since c-Myc is a potent transcriptional repressor of p21, these findings suggest that c-Myc may negatively modulate p21 expression through an additional pathway except transcriptional suppression. In the study presented here, we compared p21 mRNA in the nucleus and cytoplasm of c-Myc stable transfectants with its control, which indicated further repression of p21 by c-Myc at the post-transcriptional level. Stem-loop and conventional real-time PCR showed elevated expression of some members in miR-17 family and their primary transcripts when c-Myc was stably overexpressed. To further investigate the relationship of c-Myc, miR-17 family and p21, we antagonized the expression of each miR-17 family member by transfection of their antisense oligonucleotides respectively in transfectants constitutively overexpressing c-Myc or not, and found that the restoration of p21 expression by treatment above was much stronger in the presence of c-Myc. These results suggest c-Myc could further repress p21 expression at the post-transcriptional level through some, but not all, members of miR-17 family.

MiR-489 is a tumour-suppressive miRNA target PTPN11 in hypopharyngeal squamous cell carcinoma (HSCC)

Background:Hypopharyngeal squamous cell carcinoma (HSCC) is an aggressive malignancy with one of the worst prognoses among all head and neck cancers. Greater understanding of the pertinent molecular oncogenic pathways could help improve diagnosis, therapy, and prevention of this disease. The aim of this study was to identify tumour-suppressive microRNAs (miRNAs), based on miRNA expression signatures from clinical HSCC specimens, and to predict their biological target genes.Methods:Expression levels of 365 human mature miRNAs from 10 HSCC clinical samples were screened using stem-loop real-time quantitative PCR. Downregulated miRNAs were used in cell proliferation assays to identify a tumour-suppressive miRNA. Genome-wide gene expression analyses were then performed to identify the target genes of the tumour-suppressive miRNA.Results:Expression analysis identified 11 upregulated and 31 downregulated miRNAs. Gain-of-function analysis of the downregulated miRNAs revealed that miR-489 inhibited cell growth in all head and neck cancer cell lines examined. The gene PTPN11 coding for a cytoplasmic protein tyrosine phosphatase containing two Src Homology 2 domains was identified as a miR-489-targeted gene. Knockdown of PTPN11 resulted in the inhibition of cell proliferation in head and neck SCC cells.Conclusion:Identification of the tumour-suppressive miRNA miR-489 and its target, PTPN11, might provide new insights into the underlying molecular mechanisms of HSCC.

MiR-27a modulates MDR1/P-glycoprotein expression by targeting HIPK2 in human ovarian cancer cells

Objective MicroRNAs (miRNAs) are non-coding, single-stranded small RNAs that regulate gene expression negatively, which is involved in fundamental cellular processes and the initiation, development and progression of human cancer. In this study, we investigated the role of miR-27a in the development of drug resistance in ovarian cancer cells. Methods Expression of miR-27a in ovarian cancer cell lines A2780 and A2780/Taxol were detected by stem-loop real-time PCR. A2780 and A2780/Taxol cells were transfected with the mimics or inhibitors of miR-27a or negative control RNA (NC) by Lipofectamine 2000. The expression levels of MDR1 mRNA, P-glycoprotein (P-gp) and Homeodomain-interacting protein kinase-2 (HIPK2) proteins were assessed by real-time PCR and western blot respectively. Drug sensitivity was analyzed by MTT assay while apoptosis and the fluorescence intensity of intracellular Rhodamine 123 (Rh-123) were measured by FACS. Results The expression levels of miR-27a and P-gp were up-regulated in paclitaxel-resistant ovarian cancer cell line A2780/Taxol as compared with its parental line A2780. Transfection of A2780/Taxol cells with the inhibitors of miR-27a decreased the expression of MDR1 mRNA and P-gp protein, increased HIPK2 protein expression, enhanced the sensitivity of A2780/taxol cells to paclitaxel, increased paclitaxel-induced apoptosis and the fluorescence intensity of intracellular Rh-123. Expression of MDR1 mRNA was increased while the sensitivity to paclitaxel was decreased in A2780 cells management with the mimics of miR-27a. Conclusions The deregulation of miR-27a may be involved in the development of drug resistance, regulating the expression of MDR1/P-gp, at least in part, by targeting HIPK2 in ovarian cancer cells.

Abstract 2094: MicroRNA expression signatures in hypopharyngeal squamous cell carcinoma (HSCC): miR-489 inhibits cell proliferation by targeting PTPN11

Abstract Head and neck squamous cell carcinoma (HNSCC) affects the oral cavity, oropharynx, larynx, and hypopharynx, and is the sixth most common cancer accounting for over 500,000 new cases annually, worldwide. Hypopharyngeal squamous cell carcinomas (HSCC) are an aggressive malignancy associated with one of the worst prognoses within HNSCC. Survival rates of patients with HSCC have not improved despite recent advances in various treatment modalities, including surgery, radiotherapy and chemotherapy. Understanding of the molecular oncogenic pathways underlying HSCC would be helpful to improve diagnosis, therapy, and prevention of the disease. MicroRNAs (miRNA) are a class of naturally occurring, small, non-coding RNA that can control gene expression by targeting mRNA molecules for translational repression or cleavage. Mature miRNAs originate from pre-miRNAs that consist of 70- to 100-nucleotide hairpins. The precursors are cleaved by cytoplasmic RNase III Dicer to give around 22-nucleotide miRNA duplexes. One strand of the short-lived duplex is then degraded, whereas the other strand serves as mature miRNA. Mature miRNAs bind through partial sequence homology to the 3’-untranslated regions (3’-UTR) of target messenger RNAs, and block either translation or mRNA degradation. In cancer research, miRNAs have recently been found to be aberrantly expressed in several types of human cancer, and it appears that some miRNAs can function as oncogenes or tumor suppressors. Dysregulated miRNAs may play a role in carcinogenesis or tumor progression by altering normal gene expression patterns. The aim of this study was to identify tumor suppressive miRNAs based on miRNA expression signatures of HSCC tissues and to predict biological target genes. Expression levels of 365 human mature miRNAs were screened using the stem-loop real-time quantitative PCR method. We identified 11 up-regulated and 31 down-regulated mature miRNAs based on miRNA expression signatures. Gain-of-function analysis of down-regulated miRNAs revealed that five miRNAs (miR-489, miR-126, miR-195, miR-1 and miR-497) inhibited cell growth in cancer cell lines, with miR-489 inhibiting cell growth in all cancer cell lines examined. We identified PTPN11, a cytoplasmic protein tyrosine phosphatase that contains two Scr homology domains, as a miR-489 target gene using genome-wide gene expression analysis. miR-489-transfected cells showed reduced PTPN11 protein and mRNA expression levels. In addition, knockdown of PTPN11 expression inhibited cell growth, which suggested that PTPN11 functioned as an oncogene. Tumor suppressive miRNAs and target oncogenes may provide new insights into the mechanisms in cancer. Our findings have therapeutic implications and may be exploited for future HSCC treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2094.

Abstract 2088: miR-133a as a tumor suppressive microRNA by targeting multiple oncogenes in head and neck squamous cell carcinoma (HNSCC)

Abstract Head and neck squamous cell carcinomas (HNSCC) represent 6% of all cancers. Despite of considerable advances in surgery, radiotherapy and chemotherapy, the overall 5-year survival rate for patients is less than 40% in advanced stage. The understanding of the new molecular pathways of HNSCC carcinogenesis would be helpful in improving diagnosis and therapy of the disease. MicroRNA(miRNA) are endogenous short non-coding RNA molecules, which regulate gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. miRNA over-expression could result in down-regulation of tumor suppressor genes, whereas their under-expression could lead to oncogenes activation. Expression levels of 665 human mature miRNAs were screened using the stem-loop real-time quantitative PCR method. We identified several down-regulated miRNAs based on miRNA expression signatures in HNSCC. A comparison of miRNA signatures from HSCC and our previous reports (Ichimi et al., Int J Cancer 2009, 125:342-352; Kano et al., AACR 2009, #552; Kikkawa et al., AACR 2009, #587) identified miR-133a that is down-regulated in common, suggesting that miR-133a is candidate tumor suppressor. Gain-of-function analysis revealed that miR-133a transfectant inhibits cell proliferation and cell invasion in cancer cells. For target genes silenced by miR-133a in cancer cells, we performed genome-wide screening (oligo microarray analysis and public database searche). Among the genome-wide analysis, several candidate genes were identified, such as, fascin homolog 1 (FSCN1) and glutathione S-transferase P1 (GSTP1). We focused on FSCN1, an actin-bundling protein, which induces parallel actin bundles in cell protrusions and increases cell motility in several human cancers. The miR-133a which has conserved sequence in the 3′UTR of FSCN1, inhibited of FSCN1 expression. The signal from a luciferase reporter assay was significantly decreased at miR-133a site, suggesting miR-133a directly regulate FSCN1. An FSCN1 loss-of-function assay found significant cell growth and invasion inhibition in HNSCC cells implying an FSCN1 is associated with HSCC carcinogenesis. Tumor suppressive miRNAs and target oncogenes may provide new insights into the mechanisms in cancer. Our findings have therapeutic implications and may be exploited for future HSCC treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2088.

Hypozia Inducible Factor 1-A (HIF-1α) Upregulates MicroRNA-21 (miR-21) in Pancreatic Cancer

Background: MicroRNAs are small (∼22nt) noncoding genes capable of regulating multiple target mRNA through degradation or post-transcriptional inhibition. One of these, miR-21, has been shown to be dysregulated in a variety of cancers, including pancreatic cancer. To date, the mechanism by which miR-21 is upregulated has not been elucidated. Given that HIF-1α has a predicted binding site in the miR-21 “promoter region,” we hypothesized that hypoxia could induce miR-21 upregulation through HIF-1α expression. Methods: Human pancreatic cancer cells were grown in normoxia (21% O2) or in hypoxia (1% O2). Cells were harvested and miR-21 levels assessed by quantitative stem-loop realtime PCR. Binding between HIF-1α and miR-21 was determined by Chromatin immunoprecipitation (ChIP). Pancreatic cancer cells were then transfected with a plasmid containing a constituitively stable HIF-1 gene or empty plasmid control and miR-21 levels determined in normoxia. Results: miR-21 levels increased 8-fold in hypoxia at 72 hours but returned to baseline within 8 hours of being placed back into normoxia. ChIP analysis demonstrated that HIF-1α binds miR-21 and these levels are increased in hypoxia. Pancreatic cancer cells transfected with constituitively active HIF-1α demonstrated 3.4-fold increased miR-21 expression in normoxia relatively to cells transfected with empty vector control (p=0.004). Conclusions: Hypoxia, a potent cellular stressor, induces expression of one of the most commonly dysregulated microRNAs in cancer, miR-21. This process appears to occur through HIF-1α expression. Such adaptation potentially allows the pancreatic cancer cell to withstand the hypoxic milieu of the tumor microenvironment.

Inhibition of rabies virus replication by multiple artificial microRNAs

The RNA interference (RNAi) technology has been recognized as a promising antiviral therapy for a few years. One of the potential limitations for applying this technology against wild type rabies virus is its high rate of genetic variation. Recently, an RNAi vector system that incorporated modified dsRNA within microRNA structure [or artificial miRNAs(amiRNAs)] has been described. This allowed expression of multiple amiRNAs of single or multiple targets from a single construct. In this study, we evaluated a benefit of using amiRNA vector against different rabies strains. We found that applying single targeting amiRNA against challenged rabies virus standard (CVS) rabies nucleocapsid (N) mRNA resulted in more than 90% reduction of viral genome in Neuro2A cells up to 72 h after infection. Multiple amiRNAs aiming at single or multiple NmRNA target(s) yielded comparable inhibitory results as with a single amiRNA against perfectly matched target. Although the level of each mature miRNA generated from multiple amiRNA construct was slightly reduced as assessed by stem-loop RT and real-time PCR techniques, its effectiveness remained unchanged even when an ineffective or scrambled amiRNA was also included in the transcript. Against highly pathogenic wild type virus, single amiRNA construct activity was reduced when mismatching with target sequence occurred at critical site whereas multiple targeting amiRNA construct remained highly effective. Our results suggest the benefit of using multiple targeting amiRNAs when confronting with wild type rabies virus, the sequence of which is not completely known.

Cell-free miR-10b and miR-21 as diagnostic biomarkers for malignant effusions

11035 Background: Numerous studies have proved the roles of tissue microRNAs (miRNAs) as biomarkers in cancer diagnosis. Cell-free miRNAs from plasma or serum have been shown to be good biomarkers in the diagnosis of cancers. Here we tried to explore the potential application of cell-free miRNAs as diagnostic biomarkers to discriminate malignant effusions from benign. Methods: To obtain a molecular signature for malignant effusions, a stem-loop real-time PCR was adopted to measure the expression of 23 tumor-related miRNAs. After a pre-screening for discarding low amount miRNAs in 10 samples (5 benign/5 malignant) and a proper internal control was identified, the expression levels of 7 miRNAs were further compared among 74 malignant effusion samples (35 gastric cancer-related ascites and 39 lung cancer-related plural effusions) and 30 benign effusions. miRNAs specifically associated with malignant effusions were derived from a training group (20 malignant/10 benign) and tested in a validation group (54 malignant/20 benign). All samples were confirmed by both pathological and clinical diagnosis. Results: miR-215 was employed as the reference RNA for its abundance and consistence. The expression of miR-10b was significantly different between benign and malignant effusions (P< 0.001). Furthermore, it was showed that miR-10b can distinguish malignant plural effusions with high accuracy (areas under the curve (AUC) of receiver operating characteristic (ROC) curve 0.98, 95% CI, 0.90 to 1.0, P< 0.001), and miR-21 can be used to discriminate malignant ascites (AUC of ROC curve 0.71, 95% CI, 0.56 to 0.83, P= 0.018). Conclusions: In the present study, we demonstrated that cell-free miRNA can serve as a novel diagnostic biomarker in distinguishing malignant from benign effusions. No significant financial relationships to disclose.

Embryonic stem cells derived from somatic cloned and fertilized blastocysts are post‐transcriptionally indistinguishable: A MicroRNA and protein profile comparison

Therapeutic cloning, whereby somatic cell nuclear transfer is used to generate customized embryonic stem cells (NT-ES) from differentiated somatic cells of specific individuals, has been successfully performed in mice and non-human primates. Safety concerns have prevented this technology from being potentially applied to humans, as severely abnormal phenotypes have been observed in cloned animals. Although it has been demonstrated that the transcriptional profiles and developmental potentials of ES cells derived from cloned blastocysts are identical to those of ES cells derived from fertilized blastocysts (F-ES), a systematic analysis of the post-transcriptional profiles of NT-ES cell lines has not yet been performed. To investigate whether NT-ES cells are comparable to F-ES cells post-transcriptionally, we compared the microRNA and protein profiles of five NT- and matching F-ES cell lines by microRNA microarray, 2-D DIGE and bioinformatic analyses. Stem-loop real-time PCR and MS/MS assays were further performed to verify the expression of specific microRNAs and characterize differentially expressed proteins. Our results demonstrate that the ES cell lines derived from cloned and fertilized mouse blastocysts have highly similar microRNA and protein expression profiles, consistent with their similar developmental potentials and transcriptional profiles.

MicroRNA Signature of Primary Pigmented Nodular Adrenocortical Disease: Clinical Correlations and Regulation of Wnt Signaling

MicroRNAs comprise a novel group of gene regulators implicated in the development of different types of cancer; however, their role in primary pigmented nodular adrenocortical disease (PPNAD) has not been investigated. PPNAD is a bilateral adrenal hyperplasia often associated with Carney complex, a multiple neoplasia syndrome; both disorders are caused by protein kinase A (PKA) regulatory subunit type 1A (PRKARIA)-inactivating mutations. We identified a 44-microRNA gene signature of PPNAD after comparing PPNAD with normal adrenal samples. Specifically, 33 microRNAs were up-regulated and 11 down-regulated in PPNAD relative to normal tissues. These results were validated by stem loop real-time PCR analysis. Comparison of microRNA microarray data with clinicopathologic variables revealed a negative correlation (r = -0.9499) between let-7b expression and cortisol levels in patients with PPNAD. Integration of microRNA microarray with serial analysis of gene expression data together with bioinformatic algorithm predictions revealed nine microRNA-gene target pairs with a potential role in adrenal pathogenesis. Using a PPNAD cell line, we showed that miR-449 was up-regulated and identified its direct target, WNT1-inducible signaling pathway protein 2 (WISP2); in addition, pharmacologic inhibition of PKA resulted in the up-regulation of miR-449 leading to the suppression of WISP2. Overall, we investigated, for the first time, the microRNA profile and its clinical significance in PPNAD; these data also suggest that PKA, via microRNA regulation, affects the Wnt signaling pathway, which through expression and clinical studies is suspected to be a primary mediator of PRKAR1A-related tumorigenesis.

Detection of let-7a microRNA by real-time PCR in gastric carcinoma

To establish an accurate and rapid stem-loop reverse transcriptional real-time PCR (RT-PCR) method to quantify human let-7a miRNA in gastric cancer. According to the sequence of let-7a miRNA, the stem-loop reverse transcriptional primer, the primers and quantitative MGB probes of real-time PCR were designed and synthesized. The dynamic range and the sensitivity of quantitative reverse transcriptional real-time PCR were determined. The levels of let-7a miRNA were examined in 32 gastric carcinoma samples by stem-loop RT-PCR method. The dynamic range and sensitivity of the let-7a miRNA quantification scheme were evaluated, the result showed the assay could precisely detect 10 copies of mature let-7a miRNA in as few as 0.05 ng of total RNA of gastric mucosa. The results of specificity analysis showed no fluorescence signal occurred even though 50 ng of human genomic DNA was added to the reverse transcription (RT) reaction. The expression level of let-7a miRNA in gastric tumor tissues was significantly lower compared to normal tissues in 14 samples from 32 patients. The stem-loop RT-PCR is a reliable method to detect let-7a miRNA which may play an important role in the development of gastric carcinoma.

Distinct Expression Patterns of Human microRNAs in Myeloid Differentiation and Acute Myeloid Leukemia.

MicroRNAs (miRNAs) play an important role in differentiation, regulation of cell growth, cell death and potentially cancer pathogenesis. In the current study, we profiled expression of 154 human miRNAs by using a stem-loop reverse transcription and real-time PCR approach in 50 patients with AML and 12 control samples (normal bone marrow (NBM) samples and CD34+ cells). All of these samples were simultaneously analysed by whole genome mRNA microarrays. Out of all miRNAs tested, more than 30 were analyzed in a second, independent group of 48 AML samples and 4 controls. Five miRNAs showed differentiation associated expression changes which were confirmed by in-vitro differentiation of HL-60 cells with all-trans retinoic acid (ATRA) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Overall, we found six miRNAs, including the miR-221 and 222 cluster, that showed substantially different expression between AML and CD34+ cells/ NBM. In class prediction analyses using support vector machines, 92% of the AML samples and 100% of the control samples were correctly predicted. We also looked for differentially regulated miRNAs in FAB subtypes and identified several miRNAs with significantly different expression. For example, high expression levels of miR-26a, 23a and 27b occurred in FAB M5. mRNA levels were analyzed with regard to differences in miRNA expression. Significant negative correlations with specific mRNAs were observed for several miRNAs, for example miR-23b. When mRNA expression patterns were analyzed for dichotomized miRNA levels, close associations between specific miRNAs and gene expression clusters were observed. For example, miR-10a levels were closely associated with the expression of hox genes. Taken together, the distinct expression patterns of miRNAs observed in AML strongly suggest involvement of the described miRNAs in both hematopoietic differentiation and pathogenesis of AML.